Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Abstract

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion‐exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0.1% Triton X‐100‐treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher Km value for NADP+ than NAD+, whereas the Km value for NAD+ was higher than that for NADP+ in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42°C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N‐terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH. J. Cell. Biochem. 76:244–253, 1999. © 1999 Wiley‐Liss, Inc.

The Role of Glutamate Dehydrogenase in Mammalian Ammonia Metabolism

“…These observations agree with those derived from tissue fractionation studies (Colon et al 1986;Lee et al 1999) and ER-immunogold labeling (Aoki et al 1987) showing that GDH localizes predominantly to mitochondria and to a lesser extent so to the ER. On the other hand, Rosso et al (2008) have suggested that the signal sequence of hGDH2 shows enhanced mitochondrial targeting properties as compared to hGDH1.…”

“…This particulate-bound GDH resisted extraction by TritonX-100 and sonication, although it could be largely extracted (about 60%) by the cationic detergent CTAB and to a lesser extent (about 25%) by 0.5 M NaCl or 0.5 M KCl. Similarly, Lee et al (1999) obtained GDH from a rat liver ER fraction (105,0009g for 60 min) that was not extractable by TritonX-100, although it was partially extractable (40%) by 0.6 M NaCl. Others have reported that GDH is also located in the cell nucleus (di Prisco and Casola 1975).…”

Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

“…A recent study of chicken liver GDH [31] confirmed the nuclear localization of this enzyme [26,27,28,29] by uncovering the ability of GDH to act as a serine protease of histone H3 [31]. GDH was also found in the granular endoplasmic reticulum of rat liver [32]. Another form of GDH, which was localized in lysosomes and endosomes, has a tubulin-binding activity in the presence of ATP in vitro [33].…”

“…The existence of membrane-bound GDH forms has been shown by various researchers [24,25,27,32]. For example, a membrane-bound isoform resistant to extraction by detergents such as digitonin, deoxycholate and Triton X-100, but solubilized with cationic detergents (hexadecyltrimethylammonium bromide), and to a lesser extent with 0.5 M NaCl or KCl, is present in rat brain [27].…”

Iron alters glutamate secretion by regulating cytosolic aconitase activity

“…Indeed, in yeast, aconitase deficiency causes glutamate auxotrophy because of the lack of ␣-ketoglutarate generation (53). GDH, an enzyme with numerous isoforms in mammalian tissues (56), is found in mitochondrial and other cellular compartments, including cytosolic extracts of retina (2,10,37,57,59). GDH is present in both the retina and the retinal pigment epithelium (42).…”